This invention relates to perchlorate and nitrate metabolizing bacteria, their isolation and method of use. More particularly, this invention relates to bacteria useful in the remediation of perchlorate and nitrate contaminated materials and means and materials to enhance such remediation.
The use of bacteria to treat perchlorate and nitrate contaminated material such as soil and water is described in the xe2x80x9cDescription of Related Artxe2x80x9d of U.S. Pat. No. 6,077,429 of William T. Frankenberger, Jr. and David Herman, issued Jun. 20, 2000, entitled Bacterial Removal of Perchlorate and Nitrate, which patent is incorporated herein by reference. As discussed by Coates, Michaelidou, Bruce, O""Connor, Crespi, and Achenbach, in Ubiquity and Diversity of Dissimilatory (Per)chlorate-Reducing Bacteria, in Applied and Environmental Microbiology, December, 1999, only six organisms capable of obtaining energy for growth by the metabolism of compounds containing oxyanions of chlorine, such as perchlorates, had previously been identified at the time of Coates et al. writing, even though the use of such microbes to reduce such compounds has been known for more than fifty years. The Coates et al. article is incorporated herein by reference.
Coates et al. point out that the discovery of a phylogenitically diverse group of organisms that had evolved with the ability to couple growth to the reduction of perchlorate was unexpected. Also unexpected was the discovery of perchlorate metabolizing bacteria in environments free of perchlorates. Frankenberger et al. teach that the presence of nitrate inhibits the reduction of perchlorate by a perchlorate metabolizing bacterium.
Perchlorate is a strong oxidizing agent in its associated form and is principally manufactured as the oxidizing component in propellants and explosives. In its aqueous ionic form, the perchlorate oxyanion is extremely stable and mobile, making effective treatment difficult and expensive. It has been estimated that under typical ground and surface water conditions, the perchlorate anion may persist for decades. As the concern of perchlorate in groundwater has taken on new importance nationwide, multiple studies are currently being conducted that focus on improved analytical methods, human health assessments, ecological impact assessments, and improved treatment technologies.
It has long been known that perchlorate has the potential to perturbate the mammalian hypothalamic-pituitary-thyroid axis. Specifically, perchlorate inhibits thyroid iodide anion uptake through the action of competitive binding. This leads to reduced T3 and T4 thyroid hormones, resulting in excess Thyroid Stimulating Hormone (TSH) by the pituitary gland (Anbar et al., 1959; Stanbury and Wyngaarden, 1952; Wolff, J., 1998). Prolonged perturbations may ultimately result in thyroid neoplasia, especially in sensitive rodent species. The California Department of Health Services has adopted an action level of 18 xcexcg Lxe2x88x921 perchlorate in drinking water.
A 4.4 acre constructed wetland system, at the Apache Powder Superfund Site (APS) in Cochise County, Arizona, referred to as the Northern Area Remediation System (NARS), consisting of three primary denitrification cells, an aerobic nitrification cell, and a final denitrification cell, is intended to denitrify high levels of nitrate found in the shallow aquifer. Previous engineering design and modeling efforts for NARS did not anticipate the presence of perchlorate. Therefore, after perchlorate discovery at APS in 1998, the inventors established the current study to investigate the possibility that perchlorate would interfere with or diminish the capability of the NARS to treat nitrate containing groundwater.
Data regarding the effects of perchlorate on denitrification is generally limited. Herman and Frankenberger, Jr. (1999), using a bacterial isolate known as perclace, found a decrease in the rate of denitrification when the concentrations of nitrate and perchlorate were equal at 1 mM, requiring 48 h for complete reduction. However, when perchlorate levels were reduced to 0.089 mg Lxe2x88x921, complete nitrate reduction required only 24 h. However, Herman and Frankenberger, Jr. (1999) focused on a single bacterial isolate and not on the assemblage of microorganisms known to denitrify. One aspect of the current study focused on determining the potential effects of high levels of perchlorate on a mixed inoculant sampled from an operating wetland wastewater treatment system.
Dendooven and Anderson (1994) reported that in the presence of perchlorate, nitrous oxide production was low during the first 3-4 h, then increased sharply at 4 h and held constant for the next 20 h. After 24 h, all of the nitrate was reduced and very little nitrous oxide was produced. They suggest the lag time was due to two factors, the persistence of oxygen which delayed the de-repression of the reduction enzyme system and the kinetics of the denitrification process.
Strategies for the removal of perchlorate based on adsorption by activated carbon or use of reverse osmosis and ion exchange have not shown remediation solutions as promising as biological processes. Microorganisms utilization to degrade perchlorate in anaerobic or microaerophylic conditions to innocuous end-product, namely chloride are by far the most promising perchlorate remediation technology. Such biological treatment can be further used for the simultaneous treatment of perchlorate and nitrate. Wetlands typically contain extensive anaerobic or microaerophylic environments due to natural decomposition of plants, algae, fungi, bacteria and other organic material. Therefore, the current study also initiated preliminary treatability experiments designed to determine if enriched or non-enriched wetland derived cultures are capable of perchlorate reduction and to determine baseline kinetics.
Identified perchlorate reducers fall into several different categories. Coates et al. (1999) investigated six different environments including pristine soil, paper mill waste sludge, heavy metal contaminated aquatic sediments, hydrocarbon contaminated lake sediments, hydrocarbon contaminated soils, and animal waste treatment sludge. They recovered perchlorate reducers from all six environments. Coates et al. (1999) isolated 13 (per)chlorate reducing bacteria (CIRB), eight of which were characterized. Collectively, they represent broad phylogenetic diversities. All of the isolates were members of Proteobacteria. These bacteria were typified as being motile, gram-negative, non-fermentative, and facultative anaerobes. Their optimum growth occurred at 35xc2x0 C., pH 7.5 and 1% NaCl. All could utilize acetate, propionate, isobutyrate, butyrate, valerate, malate, fumerate and lactate as electron donors, while none could utilize methanol, catechol, glycerol, citrate, glucose or hydrogen. All of the characterized bacteria could use chlorate and oxygen as electron acceptors, but could not utilize sulfate, selenate, fumerate, malate, Mn(IV), or Fe(III). Coates et al. (1999) suggests these genera may be the dominant perchlorate reducing bacteria in the environment. Coates et al. (1999) identified and named two species of perchlorate reducers in the xcex2 sub-division of Proteobacteria, Dechlorimonas agitatus and Dechlorosoma suilla. 
In a related study, Michaelidou et al. (1999) isolated two Proteobacteria strains from a swine waste lagoon. They were both typified by being non-fermentative, mesophilic, motile, gram-negative bacteria. One strain, designated as PS, was rod-shaped and 0.2 xcexcm by 2 xcexcm in length and placed within the xcex2 sub-division of Proteobacteria. Nearly complete 16S rRNA sequencing indicated that the closest known relative was Rhodocyclus tenuis. The second strain, designated as WD, was placed into the xcex1 sub-division and shared 94.6% similarity to Magnetospirillum gryphiswaldense. Strain WD grew as a spirillum, but did not produce magnetosomes when grown in iron based media and there was no indication of magnetotaxis. Malmqvist et al. (1991) also discusses an enriched culture containing helical bacteria capable of reducing chlorate to chloride. Malmqvist et al. (1994) later describes the novel bacteria as Ideonella dechloratans, however, I. dechloratans and DM-17 share only 89% 16S rRNA gene homology.
A number of other perchlorate reducing bacteria have been studied. Rikken et al. (1996) isolated a bacteria (GR-1) from activated sludge. Sequencing of the 16S rRNA did not yield a match, but placed GR-1 in the xcex2 sub-division of Proteobacteria. The bacteria could use perchlorate, chlorate, oxygen, nitrate and Mn(IV) as electron acceptors. They also found that the GR-1 utilizes acetate, proprionate, caprionate, malate, succinate and lactate, but could not catabolize citrate, glycine, glycolate or formate. The inventors have also found that their isolate, DM-17, could not catabolize citrate. Rikken et al. (1996) also state that GR-1 was capable of completely reducing 800 mg Lxe2x88x921 of perchlorate in 9 d. The inventors found that DM-17 in static culture could completely reduce 1000 mg Lxe2x88x921 of perchlorate in 7 d.
Using a nutrient broth-yeast extract culture medium, Attaway and Smith (1993) enriched a perchlorate reducing mixed culture (gram positive cocci/rods and gram negative rods) isolated from anaerobic digester sludge. They found that the culture could use perchlorate, chlorate, chlorite, nitrate, nitrite and sulfate as electron acceptors. In contrast, Rikken et al. (1996) pointed out that GR-1 could not grow solely on chlorite because the dismutation reaction yields no energy to be used for biosynthesis. Attaway and Smith (1993) also discuss a reproducible lag time of 15-24 h prior to perchlorate reduction. The inventors"" study also showed a regular lag time of 48-72 h. However, the inventors believe the discrepancy was related to differences in initial biomass and culture medium. They found that high biomass cultures introduced into fresh media containing 10 mM perchlorate resulted in rapid and instantaneous reduction of perchlorate.
Attaway and Smith (1993) indicated that the bacteria comprising the mixed culture were strict anaerobes and that any introduction of oxygen slowed or eliminated perchlorate reduction. In fact, perchlorate reduction could only be measured when the resazurin indicator turned colorless (Eh below xe2x88x92110 mV) and once the resazurin turned pink, all perchlorate reduction ceased. Attaway and Smith (1993) suggest that transient chloride oxides such as chlorite and hypochlorite may be responsible for the oxidation of the resazurin indicator in oxygen free media. In contrast, the present study by the inventors showed that DM-17 effectively reduced perchlorate when the resazurin indicator was pink (Eh above xe2x88x92110 mV) indicating a slightly oxidized environment. Another explanation suggested by Rikken et al. (1996), is that dismutation of chlorite by GR-1 generates oxygen. Since the dismutation is the final step in converting perchlorate to chloride, it is possible that the pink color of the inventors"" cultures resulted from the dismutation of chlorite. This explanation is favored because the inventors"" cultures became colorless once perchlorate could no longer be detected by probe ( less than 10 xcexcM). This is important because the bacteria studied by Attaway and Smith (1993) were strict anaerobes and the culture medium had to maintain strictly anaerobic conditions by addition of reducing agents such as cysteine hydrochloride. In the case of bacterial isolates DM-17 and GR-1, no media manipulation was required.
Attaway and Smith (1993) also state that their cultures permanently lost the ability to reduce perchlorate when exposed to oxygen for 12 to 24 h. The DM-17 and GR-1 isolates were not deleteriously affected by the presence of oxygen, however, perchlorate reduction was temporarily inhibited. Another potential problem with the bacteria used by Attaway and Smith (1993) is the requirement for high concentrations of proteinaceous nutrients such as nutrient broth and yeast extract. They state that this requirement can be met using aged brewers yeast, cottonseed protein or whey powder. The DM-17 and GR-1 isolates do not have this requirement and can reduce perchlorate using a minimal mineral medium such as BMS with acetate or succinate serving as the carbon source.
Herman and Frankenberger, Jr. (1999) isolated the bacteria they named perclace that was found to reduce perchlorate to levels less than 0.005 mg Lxe2x88x921 when grown anaerobically on acetate. They described perclace as a gram negative, curved rod, facultative anaerobe that could reduce perchlorate or nitrate under anaerobic conditions. Gene sequencing using 16S rRNA methods indicated no similarity to any other sequenced bacteria, although they found a 90-92% sequence homology with several members of the xcex2 sub-division of Proteobacteria. Reduction of perchlorate was possible between 20-40xc2x0 C., with an optimum of 25-30xc2x0 C. Reduction of perchlorate occurred at pH 6.5-8.5, while the optimum pH was given as 7.0-7.2. These parameters closely match the optima and ranges for DM-17.
The Perlace isolate was also found to be able to use only oxygen, nitrate and perchlorate, but not Fe(III), Mn(IV), or sulfate, as electron acceptors. Using washed perclace cells, Herman and Frankenberger, Jr. (1999) found no difference in perchlorate reduction kinetics between aerobically and anaerobically grown cells. Using a 2.8 by 14 cm bioreactor column, they also found that perclace could reduce perchlorate levels below the State of California drinking water action level of 0.018 mg Lxe2x88x921. Perchlorate reduction kinetics were rapid with 580 mg Lxe2x88x921 of perchlorate reduced within a 72 h period.
Nzengung and Wang (1999) isolated four bacteria from the rhizoshere of willow trees and one was found to degrade perchlorate. The fastest degradation kinetics occurred at less than 100 mg Lxe2x88x921 nitrate-N. The degradation kinetics also decreased with increasing nitrate concentration and was attributed to competing reactions where both anions were utilized as electron acceptors. They concluded that the exposure of rooted willow trees to perchlorate containing media stimulated the growth of perchlorate reducing bacteria in the rhizoshere. This finding suggests that the NARS system may have additional modes of perchlorate reduction besides sediment localized reactions.
Perchlorate can serve as a Terminal Electron Acceptor (TEA) due to its high oxidation state (+7). Coupled to an electron donor such as acetate, perchlorate and chlorate can be fully reduced to chloride ion by bacteria grown under anaerobic and microaerophilic conditions. Rikken et al. (1996) isolated a bacteria from activated sludge belonging to the xcex2 sub-division of Proteobacteria. Rikken et al. (1996) proposed the following pathway for the reduction of perchlorate: 
Attaway and Smith (1993) used a mixed enrichment culture derived from municipal anaerobic sludge. They found protein based media provided adequate carbon sources, but simple sugars, organic acids and alcohols were inadequate for perchlorate reduction. Adequate carbon sources included nutrient broth, yeast extract, casamino acids, and peptone. Perchlorate reduction was inhibited at concentration levels higher than 77.5 mM. Attaway and Smith (1993) also showed that perchlorate reduction was inhibited by oxygen and complete and permanent inhibition occurred when the culture was subjected to 12-24 h of aeration. This suggests the principal perchlorate reducing bacteria were strict anaerobes and required redox potential (Eh) less than xe2x88x92110 mV.
Giblin et al. (1999) found that an acetate based heterotrophic bioreactor using perclace was capable of reducing 500 mg Lxe2x88x921 perchlorate to less than 5 xcexcg Lxe2x88x921 in 48 h (10.4 mg Lxe2x88x921hxe2x88x921) at 30 xc2x0 C. They also studied a hydrogen-carbon dioxide gas based autotrophic system using a consortium of 5 organisms. The autotrophic system required 96 h to reduce 500 mg Lxe2x88x921 perchlorate to less than 5 xcexcg Lxe2x88x921 (5.2 mg Lxe2x88x921hxe2x88x921) at 30xc2x0 C. Giblin et al. (1999) also found that both systems could simultaneously remove both perchlorate and 62 mg Lxe2x88x921 nitrate.
Miller and Logan (2000) demonstrated high rates of perchlorate reduction using an autotrophic (hydrogen oxidizing) packed-bed biofilm reactor. The mixed consortium autotrophic culture contained the PRB known as Dechlorosoma sp. JM. Perchlorate reduction rates averaged 13.8 mg Lxe2x88x921 hxe2x88x921 based upon a short detention time of 1.2 min. Another study using pressured hydrogen gas demonstrated a lower rate of reduction (1.02 mg Lxe2x88x921 hxe2x88x921). The JM strain could reduce perchlorate using hydrogen, but required an organic carbon source for growth. The findings of Miller and Logan (2000) suggest that no single organism can be used in a hydrogen fed autotrophic bioreactor.
Although it is believed that the enzymes responsible for perchlorate reduction are linked to nitrate reductase enzymes systems, Wallace et al. (1996) found that Wolinella succinogenes (strain HAP-1) possessed a separate perchlorate reductase enzyme system. Their reasoning was based on the observation that HAP-1 did not lose its ability to reduce perchlorate in the presence of nitrate. However, in the case of chlorate reduction, Malmquist et al. (1994) suggests that Ideonella dechloratans possesses a modified nitrate reductase enzyme system.
From the patent literature such as Frankenberger, Jr. et al., cited above, and from the journal writings such as Coates et al. (1999), it is apparent that there continues to be a present and continuing need to discover and isolate new perchlorate and nitrate reducing microorganisms and to develop processes and systems for removing perchlorates and nitrates from materials such as soil and water using such organisms. It would further be desirable to identify substances that enhance the reduction of perchlorate and nitrate by perchlorate and nitrate reducing bacteria.
In accordance with one aspect of this invention, enhanced nitrate reduction has been accomplished with a mixed bacteria culture in the presence of perchlorate. In a preferred embodiment, the mixed bacteria culture is present in a marsh sediment. Preferably, the marsh sediment is collected at the influent end of the marsh. In one particular embodiment, the marsh at which the sediment was collected was the Arcata Marsh Pilot Project, Arcata, Humboldt County, California. This sediment was collected from the upper 5 cm. of cores taken from Cell 8 at the influent end of this marsh. In one preferred method, the sediment used to denitrify a material is pretreated by exposure to perchlorate over a period of time prior to contacting the sediment with material being treated.
Unlike Dendooven and Anderson (1994), the inventors observed a marked decline in nitrous oxide after 12 h. The decline in nitrous oxide may have been due to incomplete blockage by the acetylene blocking agent, allowing nitrous oxide to further reduce to nitrogen gas. Dendooven and Anderson (1995) also found that low nitrate concentrations resulted in incomplete blockage of nitrous oxide reduction.
In accordance with another aspect of the present invention, bacteria have been isolated that are particularly useful in the treatment of materials contaminated with perchlorate and/or nitrate. One bacterium is a gram-negative, motile, polymorphic bacterium isolated from sediment collected at the influent end (Cell 3) of the Arcata Marsh Pilot Project, Arcata, Humboldt County, California. Bacteria of this form have been given the name DM-17 and have been deposited at the American Type Culture Collection, Manassas, Va., under ATCC No. PTA-2685. The bacteria DM-17 exhibits both of the unexpected qualities mentioned above, which is to say, it reduces perchlorate and grows in so doing, and it is isolated from a marsh sediment not believed to have been exposed to perchlorate.
Further, a method for the removal of perchlorate and/or nitrate from the contaminated material has been developed in accordance with this invention that includes the treatment of the material with the bacteria DM-17. Materials that enhance perchlorate remediation have been discovered to be moderate levels of nitrate, and carbon sources. Cattail and molasses have been shown to be good carbon sources for this purpose.
Although the DM-17 isolate was capable of reducing perchlorate with acetate as the sole carbon source, it was observed that the combination of organic plant material (senesced Typha latifolia leaves) and molasses yielded very high reduction kinetics. t was determined that DM-17 could reduce perchlorate at the rate of 0.18 mM hxe2x88x921 (18 mg Lxe2x88x921 hxe2x88x921) and 0.27 mM hxe2x88x921 (27 mg Lxe2x88x92hxe2x88x921), when incubated with 1 gm Lxe2x88x921 and 5 gm Lxe2x88x921 molasses, respectively. Although few studies have provided kinetic data, these rates are high in comparison to other autotrophic and heterotrophic systems.
Additionally, in accordance with the invention improved perchlorate remediation of a contaminated material includes contacting the material with the bacteria DM-17 in the presence of nitrate. Preferably, the bacteria DM-17 metabolizes both the perchlorate contaminants and nitrates in the contaminated material.
The inventors determined that the DM-17 isolate can reduce perchlorate in the presence of nitrate, but levels above 10 mM (620 mg Lxe2x88x921 nitrate or 140 mg Lxe2x88x921 nitrate-N) significantly inhibit perchlorate reduction. Other authors have discussed the need to first remove or reduce the nitrate loading of waste feeds to perchlorate bioreactors. Most studies investigating inhibitory effects of nitrate focused on levels far lower than the levels the inventors studied. For instance, Giblin et al. (2000) used 26 mg Lxe2x88x921 nitrate (equivalent to 5.9 mg Lxe2x88x921 nitrate-N) in their heterotrophic bioreactor. Herman and Frankenberger, Jr. (1999) found that perclace was unaffected when nitrate and perchlorate were equimolar. However, when the molar concentration of perchlorate was 10, 100, or 1,000 times lower than nitrate, perchlorate reduction was inhibited. The maximum nitrate concentration tested was 1 mM (62 mg Lxe2x88x921 nitrate or 5.9 mg Lxe2x88x921 nitrate-N). Herman and Frankenberger, Jr. (1999) also found that perchlorate breakthrough occurred when a bench scale sand-packed column received both 125 and 20 mg Lxe2x88x921 nitrate. Logan et al. (1999), while discussing bioreactor design considerations, points to the need to first remove nitrate from the waste stream.
Preferably, the DM-17 bacteria are used to reduce both the perchlorate and nitrate where contaminated material includes both contaminants. This ability permits the DM-17 bacteria to sustain itself in the absence of perchlorates and be effective in the reduction of perchlorate, where for example, nitrate remediation is ongoing and perchlorate appears sporadically in the material being remediated.
Also, in accordance with the present invention, a biologically pure culture of the bacteria that has been identified as DM-17 has been produced.
Although the bacteria isolated in accordance with the invention will thrive in an anaerobic environment on perchlorate, a complete absence of oxygen is not essential to perchlorate reduction.
The above and further features of the invention will be better understood with reference to the accompanying drawings taking in consideration with the following detailed description of a preferred embodiment.